High affinity tcr proteins and methods

ABSTRACT

T cell receptors (TCRS) that have higher affinity for a ligand than wild type TCRs are provided. These high affinity TCRs are formed by mutagenizing a T cell receptor protein coding sequence to generate a variegated population of mutants of the T cell receptor protein coding sequence; transforming the T cell receptor mutant coding sequence into yeast cells; inducing expression of the T cell receptor mutant coding sequence on the surface of yeast cells; and selecting those cells expressing T cell receptor mutants that have higher affinity for the peptide/MHC ligand than the wild type T cell receptor protein. The high affinity TCRs can be used in place of an antibody or single chain antibody.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/783,786, filed Feb. 20, 2004, which is a divisional of U.S.application Ser. No. 09/731,242, filed Dec. 6, 2000, which is acontinuation-in-part of U.S. application Ser. No. 09/009,388, filed Jan.20, 1998, and which claims the benefit of U.S. Provisional ApplicationNo. 60/169,179, filed Dec. 6, 1999, all of which are hereby incorporatedby reference.

ACKNOWLEDGMENT OF FEDERAL RESEARCH SUPPORT

This invention was made with Government support under Contract No.PHS-5-R01-GM55767-03 awarded by the National Institutes of Health (NIH).The Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

The field of the present invention is molecular biology, in particular,as it is related to combinatorial libraries of immune cell receptorsdisplayed on the cell surface of a recombinant host cell. Morespecifically, the present invention relates to a library of highaffinity T cell receptor proteins displayed on the surfaces ofrecombinant yeast cells, to soluble high affinity TCR receptor proteins,to high affinity TCR proteins selected for high affinity binding toparticular peptide/MHC pairs, to high affinity TCR proteins selected forbinding to a particular antigen in the absence of an MHC determinant,and to the use of the selected high affinity TCR derivatives indiagnostic methods and imaging assays, among other applications.

T cell receptors (TCRs) and antibodies have evolved to recognizedifferent classes of ligands. Antibodies function as membrane-bound andsoluble proteins that bind to soluble antigens, whereas in nature, TCRsfunction only as membrane-bound molecules that bind to cell-associatedpeptide/MHC antigens. All of the energy of the antibody:antigeninteraction focuses on the foreign antigen, whereas a substantialfraction of the energy of the TCR:peptide/MHC interaction seems to bedirected at the self-MHC molecule [Manning et al. (1998) Immunity8:413:425]. In addition, antibodies can have ligand-binding affinitiesthat are orders of magnitude higher than those of TCRs, largely becauseof the processes of somatic mutation and affinity maturation. In theirnormal cellular context, TCRs do not undergo somatic mutation, and theprocesses of thymic selection seem to operate by maintaining a narrowwindow of affinities [Alam et al. (1996) Nature 381:616-620]. Theassociation of TCRs at the cell surface with the accessory molecules CD4or CD8 also may influence the functional affinity of TCRs [Garcia et al.(1996) Nature 384:577-581]. Despite these differences, thethree-dimensional structures of the two proteins are remarkably similar,with the hypervariable regions forming loops on a single face of themolecule that contacts the antigen.

Based on their structural similarities, it is somewhat surprising thatthere have been significant differences in the success of producingsoluble and surface-displayed forms of the extracellular domains of TCRsand antibodies in heterologous expression systems. Many antibodies havenow been expressed at high yield and solubility as either intact orFab-fragment forms or as single-chain (sc) fragment-variable (Fv)proteins. In addition, there are numerous antigen-binding Fv fragmentsthat have been isolated de novo and/or improved through the use ofphage-display technology and, more recently, with yeast-displaytechnology [Boder and Wittrup (1997) Nat. Biotechnol. 15:553-557; Kiekeet al. (1997) Prot. Eng. 10:1303-1310]. These expression systems forantibody fragments have been key in structural studies and in the designof diagnostic and therapeutic antibodies.

In contrast, the three-dimensional structures of a few TCR moleculeswere determined only after considerable effort on the expression ofsoluble, properly folded TCRs [Bentley and Mariuzza (1996) Ann. Rev.Immunol. 14:563-590]. One of the difficulties in exploring the basis ofdifferences between Fab and TCR has been that the extensive sequencediversity in antibody and TCR variable (V) regions complicates effortsto discern what features of the V regions are important for functionsother than antigen binding (e.g., V region pairing and associationkinetics, stability, and folding). There have been relatively fewstudies that have compared the V regions of TCRs and antibodies in termsof these properties.

Nevertheless, the TCR from the mouse T cell clone 2C has now beenexpressed as an sc V_(α)V_(β) (scTCR) in Escherichia coli [Soo Hoo etal. (1992) Proc. Natl. Acad. Sci. USA 89:4759-4763], as a lipid-linkedV_(α)C_(α)V_(β)C_(β) dimer from myeloma cells [Slanetz and Bothwell(1991) Eur. J. Immunol. 21:179-183], and as a secretedV_(α)C_(α)V_(β)C_(β) dimer from insect cells [Garcia et al. (1996)Science 274:209-219]. The 2C scTCR had relatively low solubilitycompared with most scFv, although its solubility is increased about10-fold by fusion at the amino terminus to thioredoxin [Schodin et al.(1996) Molec. Immunol. 33:819-829]. The difficulty in generatingsoluble, properly folded V_(α)V_(β) domains has extended to other TCRs[Udaka et al. (1993) supra; Sykulev et al. (1994) supra; Manning et al.(1998) supra]. The molecular explanation for the apparent differencesbetween TCR and Fv in either solubility or surface-display capabilityhas not been explored adequately. It has been shown that the 2C scTCRcan be expressed in a yeast surface-display system after the selection,from a random library, of specific single-site mutations at theV_(α)/V_(β) interface or in a region of the V_(β) framework suspected tointeract with the CD3, signal-transduction sub-unit. These mutations,several of which are found naturally in antibody V regions, reflect thesignificance of these positions in the TCR and provide a basis forfurther engineering of TCR-binding properties.

SUMMARY OF THE INVENTION

The invention provides a combinatorial library of immune T cell receptorpolypeptides displayed on the surfaces of recombinant host cells, forexample, yeast cells, desirably Saccharomyces cerevisiae. From such alibrary can be isolated high affinity TCR polypeptides (those thatexhibit higher affinity than wild type for the cognate ligand: a complexof peptide bound to a protein of the major histocompatibility complex,pMHC). Desirably, the affinity of the TCR peptide for the pMHC isreflected in a dissociation constant of from about 10⁷ to about 10¹⁰,e.g., as measured by methods known to the art. A DNA library comprisingnucleic acids encoding soluble high affinity TCRs, wherein said TCRs aremade by the method of mutagenizing a TCR to create mutant TCR codingsequences; transforming DNA comprising the mutant TCR coding sequencesfor mutant TCRs into yeast cells; inducing expression of the mutant TCRcoding sequences such that the mutant TCRs are displayed on the surfaceof yeast cells; contacting the yeast cells with a fluorescent labelwhich binds to the peptide/MHC ligand to produce selected yeast cells;and isolating the yeast cells showing the highest fluorescence isprovided. Also provided is a library of T cell receptor proteinsdisplayed on the surface of yeast cells which have higher affinity forthe peptide/MHC ligand than the wild type T cell receptor protein,wherein said library is formed by mutagenizing a T cell receptor proteincoding sequence to generate a variegated population of mutants of the Tcell receptor protein coding sequence; transforming the T cell receptormutant coding sequence into yeast cells; inducing expression of the Tcell receptor mutant coding sequence on the surface of yeast cells; andselecting those cells expressing T cell receptor mutants that havehigher affinity for the peptide/MHC ligand than the wild type T cellreceptor protein.

The present invention further provides TCR proteins (in cell-bound or insoluble form) that exhibit high affinity binding for the cognate ligand.In the present invention the ligand bound by the TCR protein can be apeptide/MHC complex or because of the selection process, desirably aniterated selection process, it can be a ligand which does not include anMHC component, such as a superantigen. This ligand can be a peptide, aprotein, a carbohydrate moiety, or a lipid moiety, among others. Thesesoluble high affinity TCRs may be made by the method comprising:mutagenizing a TCR to create mutant TCR coding sequences; transformingDNA comprising the mutant TCR coding sequences for mutant TCRs intoyeast cells; inducing expression of the mutant TCR coding sequences suchthat the mutant TCRs are displayed on the surface of yeast cells;contacting the yeast cells with a fluorescent label which binds to thepeptide/MHC ligand to produce selected yeast cells; and isolating theyeast cells showing the highest fluorescence. The soluble high affinityTCRs are preferably isolated by yeast display.

The present invention further provides methods for detecting the cognateligand of a high affinity TCR protein, said methods comprising the stepof binding the high affinity TCR protein with the cognate ligand, wherethe high affinity TCR protein is detectably labeled or where there is asecondary detectable protein added, such as an antibody specific for theTCR in a region other than the region which binds the cognate ligand. Apreferred method for using high affinity TCRs to identify ligandscomprises: labeling high affinity TCRs with a detectable label;contacting said labeled TCRs with ligands; identifying the ligand withwhich the labeled TCR is bound. Preferably the ligands are thosepeptide/MHC ligands to which the TCR binds with higher affinity than thewild type TCR. Methods of identifying the ligand are known to one ofordinary skill in the art. Suitable labels allowing for detection of theTCR protein, directly or indirectly, include but are not limited tofluorescent compounds, chemiluminescent compounds, radioisotopes,chromophores, and others.

The high affinity TCR protein can be used in the laboratory as a toolfor qualitative and quantitative measurements of a target ligand, inmedical, veterinary or plant diagnostic setting or for tissue or plantmaterial identification. Similarly, the high affinity TCRs of thepresent invention can be used as reagents for detecting and/orquantitating a target material or ligand. Also provided is a method ofusing high affinity TCRs to bind to a selected peptide/MHC ligandcomprising: labeling said high affinity TCRs with a label that binds tothe selected peptide/MHC ligand; contacting said labeled high affinityTCRs with cells containing MHC molecules. The high affinity protein ofthe present invention, where it specifically binds to a tumor cellantigen with high affinity and specificity can be used in diagnostictests for the particular type of cancer or it can be used in an organismin imaging tests to locate and/or estimate size and number of tumors inan organism, preferably a mammal, and also preferably a human. Methodsprovided for using high affinity TCRs that bind to pMHCs for diagnostictests comprise: labeling the high affinity TCR with a detectable label;contacting said high affinity TCR with cells containing the ligand towhich the high affinity TCR has high affinity for; and detecting thelabel. In the method, the label may be chosen to bind to specificpeptide/MHC ligands, whereby cells that express specific peptide/MHCligands are targeted. Preferred methods for using high affinity TCRs asdiagnostic probes for specific peptide/MHC molecules on surfaces ofcells comprise: labeling high affinity TCRs with a detectable label thatbinds to specific peptide/MHC ligands; contacting said TCRs with cells;and detecting said label. The detectable label chosen for use depends onthe particular use, and the choice of a suitable label is well withinthe ordinary skill of one in the relevant art. In general, the TCRproteins selected for high affinity binding to a ligand of interest canbe used in methods in which antibodies specific for the ligand can beused, with procedural modifications made for the TCR vs. antibodyprotein, such modifications being known in the art.

The high affinity TCR, desirably a soluble single chain (sc) TCR, can beused to block autoimmune destruction of cells or tissues in autoimmunedisease, where the site recognized by the cytotoxic lymphocytes on thesurface of the target cell is the same as the site bound by the highaffinity TCR. Preferred methods for blocking autoimmune destruction ofcells comprise contacting TCRs with high affinity for the siterecognized by the T lymphocytes on the surface of a target cell withcells, whereby the autoimmune destruction of cells is blocked.

A soluble, high affinity scTCR can be coupled to a therapeutic compound(e.g., an anticancer compound, a therapeutic radionuclide or a cytoxicprotein) where the cognate ligand of the sc TCR is a neoplastic cellsurface marker. Alternatively, the binding specificity of the highaffinity soluble sc TCR can be a pathogen infected target cell (such asvirus-, bacteria- or protozoan-infected) and a toxic molecule can becoupled so that the target cell can be eliminated without furtherreplication of the infective agent. Provided methods of using highaffinity TCRs to inactivate pathogens comprise: binding a molecule whichis toxic to the pathogen to the high affinity TCR; and contacting saidTCR with cells that express said pathogen. “Toxic” means that thepathogen prevents or inhibits replication of the pathogen.

Also provided are methods for using high affinity TCRs to treat diseasecomprising: coupling a TCR having a high affinity for a neoplastic cellsurface marker with a therapeutic compound; and contacting said TCR withcells. Any therapeutic compound that is useful in slowing the progressof the disease that can be coupled with the TCR may be used. Methods ofcoupling the therapeutic compound with the TCR are known in the art.

Also provided is a method for cloning the gene for a high affinity TCRmutant into a system that allows expression of the mutant on the surfaceof T cells comprising: mutating TCRs to create high affinity TCRmutants; cloning said TCR mutants into a vector; transfecting the vectorinto T cells; expressing the high affinity TCR mutant on the surface ofT cells. This method may further comprise: selecting those T cells thatare activated to a greater extent than other T cells by a peptide/MHCligand. The transfected/infected T cells may be used for recognition ofselected peptide-bearing MHC cells. These transfected/infected T cellsare useful in treating disease in patients where T cells from a patientare removed and transformed with the vector that expresses the highaffinity TCR mutants and returned to the patient where they areactivated to a greater extent by a peptide/MHC ligand than the patient'swild type T cells.

A soluble, high affinity TCR molecule can be used in place of anantibody or single chain antibody for most applications, as will bereadily apparent to one of skill in the relevant arts.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Flow cytometric analysis of yeast cells that express wild-typeand mutant 2C TCR on their surfaces. Yeast cells displaying wild-type(T7) and mutant (qL2, qL7) scTCR were stained with anti-Vβ8 antibodyF23.2 (120 nM), the specific alloantigenic peptide-MHC, QL9/L^(d)/Ig (40nM), or a null peptide MCMV (SEQ ID NO: 1)/L^(d)/Ig (40 nM). Binding wasdetected by FITC-conjugated goat anti-mouse IgG F(ab′)₂ and analyzed byflow cytometry. The negative population (e.g. seen with F23.2 staining)has been observed for all yeast displayed-proteins and is thought to bedue to cells at a stage of growth or induction that are incapable ofexpressing surface fusion protein (Kieke et al. (1999) Proc. Natl. Acad.Sci. USA 96:5651-5656; Boder and Wittrup (1997) Nat. Biotech.15:553-557; Kieke et al. (1997) Protein Engineering 10:1303-1310).

FIG. 2: Fine specificity analysis of mutant scTCR binding to differentQL9 variant peptides bound to L^(d). The original T cell clone 2C andvarious yeast clones were analyzed by flow cytometry for binding toL^(d)/Ig dimers loaded with wild type QL9 (P5F), position 5 variants ofQL9 (P5Y, P5H, P5E) or MCMV (SEQ ID NO: 1). Binding was detected withFITC-labeled goat anti-mouse IgG. Relative fluorescence was measured bytwo different approaches. For T cell clone 2C, the binding of thevarious peptide/L^(d) Ig dimers was adjusted relative to the QL9/L^(d)staining (MFU_(pMHC)/MFU_(QL9-Ld)). For yeast cells, the binding of eachpeptide/L^(d) dimer was adjusted relative to binding by the anti-Vβ8antibody F23.2 (MFU_(pMHC)/MFU_(F23.2)). The latter allowed differentmutants to be compared relative to each other for binding to the wildtype QL9/L^(d).

FIG. 3: QL9/L^(d) binding by soluble scTCRs. T2-L^(d) cells loaded withQL9 were incubated with ¹²⁵I-labeled anti-L^(d) Fab fragments (30-5-7)and various concentrations of unlabeled Fab (♦), scTCR-T7 (▪), or mutantscTCR-qL2 (). Bound and unbound ¹²⁵I 30-5-7 Fab fragments wereseparated by centrifugation through olive oil/dibutyl phthalate. Bindingof ¹²⁵I-labeled anti-L^(d) Fab fragments to T2-L^(d) cells loaded withthe control peptide MCMV (SEQ ID NO: 1) was not inhibited even at thehighest concentrations of scTCRs (data not shown).

FIG. 4: Flow cytometric analysis of the binding of scTCR/biotin to cellsurface peptide/MHC. Peptide-loaded T2-L^(d) cells were incubated withbiotinylated qL2 scTCR (˜0.3 μM) or T7 scTCR (˜1.6 μM) scTCR followed bystreptavidin-PE and analyzed by flow cytometry. FIG. 4A: Flow cytometryhistograms of T2-L^(d) cells loaded with QL9 (unshaded), p2Ca (lightshade), or MCMV (SEQ ID NO: 1) (dark shade) and stained with qL2scTCR/biotin. FIG. 4B: Mean fluorescent units (MFU) of T2-L^(d) cellsloaded with QL9, p2Ca, or MCMV (SEQ ID NO: 1) and stained with eithersecondary SA-PE only, T7 scTCR/biotin+SA-PE, or qL2 scTCR/biotin+SA-PE.FIG. 4C: A soluble, high affinity form of mutant qL2 expressed frominsect cells can detect very low concentrations of a peptide-MHCcomplex. Ld complexes were up-regulated on the surface of T2-Ld cells(3×10⁶/ml) by incubation with various concentrations of QL9 peptide at37° C. for 1.5 hr. Approximately 2×10⁵ cells were stained for 30 min onice with TCRs derived from transfected Drosophila melanogaster (insect)SC2 cells (Garcia, K. C., et al. (1997) Proc Natl Acad Sci USA 94(25),13838-13843). Cells were then washed and stained with biotin-labeledanti-Vb IgG (F23.1) followed by streptavidin-PE and analyzed by flowcytometry.

FIG. 5: Flow cytometry histograms of yeast displaying a mutant scTCR(called 3SQ2) stained with biotinylated peptide/MHC complexes,OVA/K^(b), dEV8/K^(b) or SIYR (SEQ ID NO:2)/K^(b), followed bystreptavidin-PE. As a positive control for the presence of scTCR, yeastwere stained with the Vβ-specific Ig, F23.2 followed by FITCgoat-anti-mouse F(ab′)₂.

FIG. 6: T2-K^(b) tumor cells were incubated with specific peptides (OVA,dEV8 or SIYR (SEQ ID NO:2)) and analyzed by flow cytometry staining withbiotinylated soluble scTCR, 3SQ2 followed by streptavidin-PE. As apositive control for the presence of K^(b), T2-K^(b) cells were stainedwith biotinylated antibody B8.24.3, which recognizes K^(b) irrespectiveof the bound peptide.

FIG. 7: After multiple rounds of sorting with dEV8/K^(b), the yeastVαCDR3 library was stained with biotinylated dEV8/K^(b) followed bystreptavidin-PE and analyzed by flow cytometry.

FIG. 8: Flow cytometry histograms of yeast displaying a mutant scTCR(called 4d1) stained with biotinylated peptide/MHC complexes, OVA/K^(b)or dEV8/K^(b) followed by staining with biotinylated streptavidin-PE. Aspositive controls the yeast were analyzed for the presence of scTCR Vβwith F23.2 Ig and for epitope tags with an anti-6His antibody or theanti-HA Ig, 12CA5.

FIG. 9: T cells transfected with the mutant T cell receptor qL2 canrecognize and be stimulated by target cells that express the peptide-MHCat low concentrations. T-cell hybridoma cell line 58−/− (Letoumeur, F.,and B. Malissen. (1989) Eur J Immunol 19(12), 2269-74) was transfectedwith the wild-type (2C) or mutant (qL2) TCRs and 7.5×10⁴ transfectedcells/well were incubated at 37° C. with T2-Ld cells (7.5×10⁴/well) inthe presence of QL9 peptide. After ˜30 hrs, supernatants were collectedand assayed for IL-2 released by the T cells: Supernatants wereincubated with the IL-2 dependent cell line, HT2 (5×10³/well) for 18 hrsat 37° C. Proliferation of HT2 cells was measured by the incorporationof 3 [H] thymidine. Mean CPM represents the average of triplicate wells.No IL-2 was released in the absence of the QL9 peptide (data not shown).

FIG. 10: Structure and sequences of the 2C TCR CDR3α. Alignment of aminoacid sequences of mutant scTCRs isolated by yeast display and selectionwith QL9/L^(d). Display plasmids were isolated from yeast clones afterselection and sequenced to determine CDR3α sequences. Mutants m1, m2,m3, m4, m10 and m11 were isolated after the third round of sorting. Allother mutants were isolated after the fourth round of sorting.

FIG. 11: SIYR/K^(b) Binders (3SQ2, 3SQ5): CDR3α Sequences.

FIG. 12: Alignment of VαCDR3 Mutant Sequences with High Affinity fordEV8/Kb (4d1, 4d2, 3Sd3, 3dS6, 3dS2, 3d2) and SIYR/K^(b).

FIG. 13: Alignment of VβCDR3 Sequences of Mutant scTCRs Selected forHigh Affinity for QL9/L^(d) from a CDR3α Yeast Library. All have qL2CDR3α (SHQGRYL (SEQ ID NO: 13)). QB1/5=wt; QB3 not sequenced.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given to such terms,the following definitions are provided.

A coding sequence is the part of a gene or cDNA which codes for theamino acid sequence of a protein, or for a functional RNA such as a tRNAor rRNA.

Complement or complementary sequence means a sequence of nucleotideswhich forms a hydrogen-bonded duplex with another sequence ofnucleotides according to Watson-Crick base-pairing rules. For example,the complementary base sequence for 5′-AAGGCT-3′ is 3′-TTCCGA-5′.

Downstream means on the 3′ side of any site in DNA or RNA.

Expression refers to the transcription of a gene into structural RNA(rRNA, tRNA) or messenger RNA (mRNA) and subsequent translation of amRNA into a protein.

An amino acid sequence that is functionally equivalent to a specificallyexemplified TCR sequence is an amino acid sequence that has beenmodified by single or multiple amino acid substitutions, by additionand/or deletion of amino acids, or where one or more amino acids havebeen chemically modified, but which nevertheless retains the bindingspecificity and high affinity binding activity of a cell-bound or asoluble TCR protein of the present invention. Functionally equivalentnucleotide sequences are those that encode polypeptides havingsubstantially the same biological activity as a specifically exemplifiedcell-bound or soluble TCR protein. In the context of the presentinvention, a soluble TCR protein lacks the portions of a nativecell-bound TCR and is stable in solution (i.e., it does not generallyaggregate in solution when handled as described herein and understandard conditions for protein solutions).

Two nucleic acid sequences are heterologous to one another if thesequences are derived from separate organisms, whether or not suchorganisms are of different species, as long as the sequences do notnaturally occur together in the same arrangement in the same organism.

Homology refers to the extent of identity between two nucleotide oramino acid sequences.

Isolated means altered by the hand of man from the natural state. If an“isolated” composition or substance occurs in nature, it has beenchanged or removed from its original environment, or both. For example,a polynucleotide or a polypeptide naturally present in a living animalis not isolated, but the same polynucleotide or polypeptide separatedfrom the coexisting materials of its natural state is isolated, as theterm is employed herein.

A linker region is an amino acid sequence that operably links twofunctional or structural domains of a protein.

A nucleic acid construct is a nucleic acid molecule which is isolatedfrom a naturally occurring gene or which has been modified to containsegments of nucleic acid which are combined and juxtaposed in a mannerwhich would not otherwise exist in nature.

Nucleic acid molecule means a single- or double-stranded linearpolynucleotide containing either deoxyribonucleotides or ribonucleotidesthat are linked by 3′-5′-phosphodiester bonds.

Two DNA sequences are operably linked if the nature of the linkage doesnot interfere with the ability of the sequences to effect their normalfunctions relative to each other. For instance, a promoter region wouldbe operably linked to a coding sequence if the promoter were capable ofeffecting transcription of that coding sequence.

A polypeptide is a linear polymer of amino acids that are linked bypeptide bonds.

Promoter means a cis-acting DNA sequence, generally 80-120 base pairslong and located upstream of the initiation site of a gene, to which RNApolymerase may bind and initiate correct transcription. There can beassociated additional transcription regulatory sequences which provideon/off regulation of transcription and/or which enhance (increase)expression of the downstream coding sequence.

A recombinant nucleic acid molecule, for instance a recombinant DNAmolecule, is a novel nucleic acid sequence formed in vitro through theligation of two or more nonhomologous DNA molecules (for example arecombinant plasmid containing one or more inserts of foreign DNA clonedinto at least one cloning site).

Transformation means the directed modification of the genome of a cellby the external application of purified recombinant DNA from anothercell of different genotype, leading to its uptake and integration intothe subject cell's genome. In bacteria, the recombinant DNA is nottypically integrated into the bacterial chromosome, but insteadreplicates autonomously as a plasmid.

Upstream means on the 5′ side of any site in DNA or RNA.

A vector is a nucleic acid molecule that is able to replicateautonomously in a host cell and can accept foreign DNA. A vector carriesits own origin of replication, one or more unique recognition sites forrestriction endonucleases which can be used for the insertion of foreignDNA, and usually selectable markers such as genes coding for antibioticresistance, and often recognition sequences (e.g. promoter) for theexpression of the inserted DNA. Common vectors include plasmid vectorsand phage vectors.

High affinity T cell receptor (TCR) means an engineered TCR withstronger binding to a target ligand than the wild type TCR.

T cells recognize a foreign peptide bound to the MHC product through theαβ heterodimeric T cell receptor (TCR). The TCR repertoire has extensivediversity created by the same gene rearrangement mechanisms used inantibody heavy and light chain genes [Tonegawa, S. (1988) Biosci. Rep.8:3-26]. Most of the diversity is generated at the junctions of variable(V) and joining (J) (or diversity, D) regions that encode thecomplementarity-determining region 3 (CDR3) of the α and β chains [Davisand Bjorkman (1988) Nature 334:395-402]. However, TCRs do not undergosomatic point mutations as do antibodies and, perhaps notcoincidentally. TCRs also do not undergo the same extent of affinitymaturation as antibodies. TCRs as they occur in nature appear to haveaffinities that range from 10⁵ to 10⁶ M⁻¹ whereas antibodies typicallyhave affinities that range from 10⁵ to 10⁹ M⁻¹ [Davis et al. (1998)Annu. Rev. Immunol. 16:523-544; Eisen et al. (1996) Adv. Protein Chem.49:1-56]. While the absence of somatic mutation in TCRs may beassociated with lower affinities, it has also been argued that there isnot a selective advantage for a TCR to have higher affinity. In fact,the serial-triggering [Valitutti et al. (1995) Nature 375:148-151] andkinetic proofreading [Rabinowitz et al. (1996) Proc. Natl. Acad. Sci.USA 93:1401-1405] models of T cell activation both suggest that longeroff-rates (associated with higher affinity) would be detrimental to thesignaling process. It is also possible that higher affinity TCRs mightnot maintain the peptide specificity required for T cell responses. Forexample, peptides bound within the MHC groove display limited accessiblesurface [Bjorkman, P. J. (1997) Cell 89:167-170], which may in turnlimit the amount of energy that can be generated in the interaction. Onthe other hand, raising the affinity of a TCR by directing the energytoward the MHC helices would presumably lead to thymic deletion duringnegative selection [Bevan, M. J. (1997) Immunity 7:175-178].

We show that there is not an inherent structural property or geneticlimitation on higher affinity of T cell receptor proteins. Higheraffinity TCR variants were generated in the absence of in vivo selectionpressures by using yeast display combinatorial technology and TCRmutants (e.g., Vα and Vβ CDR3 mutants). Mutants selected for relativelystrong binding to the target ligand (a particular p/MHC complex) canhave greater than 100-fold higher affinity, i.e., a K_(d) of about 10 nMfor the p/MHC, and these mutants retained a high degree of peptidespecificity. A strong preference for TCR proteins with conserved CDR3motifs that were rich in proline or glycine were also evident. A solublemonomeric form of a high affinity TCR was capable of directly detectingp/MHC complexes on antigen-presenting cells. These findings prove thataffinity maturation of TCRs is possible, at least in vitro. Thus,engineered TCR proteins can be used for targeting specific ligands,including particular p/MHC complexes and peptides, proteins or otherligands in the absence of a MHC component.

To examine if it is possible to generate higher affinity TCR that retainpeptide specificity, we subjected a characterized TCR to a process ofdirected in vitro evolution. Phage display technology [Clackson et al.(1991) Nature 352:624-628] has not yet proven successful in theengineering of single-chain TCRs (scTCRs, Vβ-linker-Vα), despite theextensive structural similarity between antibody and TCR V regions.However, we recently showed that a scTCR could be displayed on thesurface of yeast [Kieke et al. (1999) Proc. Natl. Acad. Sci. USA96:5651-5656], in a system that has proven successful in antibodyengineering [Boder and Wittrup (1997) supra; Kieke et al. (1997) supra].A temperature-stabilized variant (called T7) [Shusta et al. (1999) J.Mol. Biol. 292:949-956] of the scTCR from the CTL clone 2C was used inthe present study. CTL clone 2C recognizes the alloantigen Ld with abound octamer peptide called p2Ca, derived from the enzyme2-oxoglutarate dehydrogenase [Udaka et al. (1993) Proc. Natl. Acad. Sci.USA 90:11272-11276]. The nonameric variant QL9 is also recognized by CTL2C, but with 10-fold higher affinity by the 2C TCR [Sykulev et al.(1994) Proc. Natl. Acad. Sci. USA 91:11487-11491]. Alanine scanningmutagenesis shows that the CDR3α loop contributed minimal energy to thebinding interaction [Manning (1998) supra], even though structuralstudies have shown that CDR3α of the 2C TCR is near the peptide and itundergoes a conformational change in order to accommodate the pMHCcomplex [Garcia (1998) Science 279:1166-1172]. Thus, we focused ourmutagenesis efforts on the five residues that form the tip of CDR3.

A library of 10⁵ independent TCR-CDR3α yeast mutants was subjected toselection by flow cytometry with a fluorescently-labeled QL9/L^(d)ligand [Dal Porto et al. (1993) Proc. Natl. Acad. Sci. USA90:6671-6675]. After four rounds of sorting and growth, fifteendifferent yeast colonies were examined for their ability to bind theligand, in comparison to the scTCR variant T7, which bears the wt CRD3αsequence (FIG. 1). The anti-Vβ8.2 antibody F23.2 which recognizesresidues in the CDR1 and CDR2 regions of the protein was used as acontrol to show that wt scTCR-T7 and scTCR mutants (qL2 and qL7 in FIG.1 and others) each had approximately equivalent surface levels of thescTCR (FIG. 1). In contrast, the soluble QL9/L^(d) ligand bound verywell to each mutant yeast clone but not to wt scTCR-T7. The MCMV (SEQ IDNO: 1)/L^(d) complex, which is not recognized by CTL clone 2C, did notbind to the scTCR mutants or to the wt scTCR-T7, indicating that thescTCR mutants retained peptide specificity. The relative affinities ofthe mutant TCR also appeared to vary among clones, based on differencesin signals observed with the QL9/L^(d) ligand at constantconcentrations.

CDR3α sequences of the fifteen mutants all differed from the starting 2CTCR sequence (FIG. 10). Comparison by a BLAST alignment algorithmaligned the sequences into two motifs. One motif contained glycine inthe middle of the 5 residue stretch whereas the other motif containedthree tandem prolines. Evidence that all three prolines are important ingenerating the highest affinity site is suggested by results with mutantq3r. Mutant q3r contained only two of the three prolines and exhibitedreduced binding compared to the triple-proline mutants. Theglycine-containing mutants appeared to have preferences forpositive-charged residues among the two residues to the carboxy side(7/9) and aromatic and/or positive-charged residues among the tworesidues to the amino side (4/9 and 5/9). Without wishing to be bound bytheory, it is believed that the selection for a glycine residue atposition 102 in the motif indicates that the CDR3α loop requiresconformational flexibility around this residue in order to achieveincreased affinity. This is consistent with the large (6 Å)conformational difference observed between the CDR3α loops of theliganded and unliganded TCR [Garcia et al. (1998) supra]. It is alsointeresting to note that glycine is the most common residue at the V(D)Jjunctions of antibodies and that the presence of a glycine has recentlybeen associated with increased affinity in the response to the(4-hydroxy-3-nitrophenyl)acetyl hapten [Furukawa et al. (1999) Immunity11:329-338].

In contrast to the isolates that contain glycine, the selection for aproline-rich sequence at the tip of the CDR3α loop is believed, withoutwishing to be bound by any particular theory, to indicate that these TCRmolecules exhibit a more rigid conformation that confers higheraffinity. The X-ray crystallographic structures of a germ line antibodyof low affinity compared to its affinity-matured derivative showed thatthe high affinity state may have been due to the stabilization of theantibody in a configuration that accommodated the hapten [Wedemayer etal. (1997) Science 276:1665-1669]. Similarly, the NMR solution structureof a scTCR that may be analogous to the germline antibody showed thatthe CDR3α and β loops both exhibited significant mobility [Hare et al.(1999) Nat. Struct. Biol. 6:574-581]. Recent thermodynamic studies ofTCR:pMHC interactions have also suggested the importance ofconformational changes in binding [Willcox et al. (1999) Immunity10:357-365; Boniface et al. (1999) Proc. Natl. Acad. Sci. USA96:11446-11451]. Structural and thermodynamic studies of the TCR mutantsdiscussed herein allowed us to examine if the two CDR3α motifs (Glyversus Pro-rich) differ in the mechanism by which they confer higheraffinity.

Although the scTCR mutants did not bind the null (irrelevant) peptide/Ldcomplex MCMV (SEQ ID NO:1)/L^(d), it remained possible that the increasein affinity was accompanied by a change in fine specificity. To examinethis question, we used QL9 position 5 (Phe) peptide variants which havebeen shown previously to exhibit significant differences in theirbinding affinity for the wt 2C TCR [Schlueter (1996) J. Immunol.157:4478-4485]. The binding of these pMHC to various TCR mutants on theyeast surface and to clone 2C was measured by flow cytometry. As shownin FIG. 2, the native TCR on 2C is capable of binding QL9 variants thatcontain either tyrosine or histidine at position 5 but not thosecontaining glutamic acid. Each of the higher affinity TCR mutantsretained the ability to recognize the conservative tyrosine-substitutedpeptide, and they were likewise incapable of recognizing the glutamicacid-substituted peptide. However, several of the TCR mutants (qL2, qL5,and qL7) bound to the histidine-substituted peptide (albeit to differentextents) whereas other mutants (qL1, qL3, and qL8) did not bind thispeptide (within the detection limits of this assay). Thus, the CDR3αloop can influence the peptide fine specificity of recognition, but itis not the only region of the TCR involved. The effect on peptidespecificity could be through direct interaction of CDR3α residues withthe variant peptide, as suggested from earlier studies involvingCDR3-directed selections [Sant'Angelo et al. (1996) Immunity 4:367-376;Jorgensen et al. (1992) Nature 355:224-230]. Alternatively, bindingenergy may be directed at peptide-induced changes in the L^(d) moleculeitself. The latter possibility is perhaps more likely in the case of the2C TCR:QL9/L^(d) interaction, as position 5 of QL9 has been predicted topoint toward the L^(d) groove [Schlueter et al. (1996) supra; Speir etal. (1998) Immunity 8:553-562]. The fine-specificity analysis also showsthat it is possible to engineer TCR with increased, or at least altered,specificity for cognate peptides. Thus, directed evolution of only ashort region (CDR3α) of a single TCR allows the isolation of many TCRvariants with desirable peptide-binding specificities and/or increasedbinding affinities.

In order to determine the magnitude of the affinity increases associatedwith a selected CDR3α mutant, the wild type T7 scTCR and the qL2 scTCRwere expressed as soluble forms in a yeast secretion system. PurifiedscTCR preparations were compared for their ability to block the bindingof a ¹²⁵I-labeled anti-L^(d) Fab fragments to QL9 or MCMV (SEQ ID NO: 1)loaded onto Ld on the surface of T2-L^(d) cells [Manning (1998) supra;Sykulev et al. (1994) Immunity 1: 15-22]. As expected, neither T7 norqL2 scTCR were capable of inhibiting the binding of ¹²⁵I-Fab fragmentsto T2-L^(d) cells upregulated with the MCMV (SEQ ID NO: 1) peptide.However, both T7 and qL2 were capable of inhibiting the binding ofanti-L^(d) Fab fragments to QL9/L^(d) (FIG. 3). The qL2 scTCR variantwas as effective as unlabeled Fab fragments in inhibiting binding,whereas the T7 scTCR was 160-fold less effective (average of 140-folddifference among four independent titrations). The K_(D) values of thescTCR for the QL9/L^(d) were calculated from the inhibition curves to be1.5 μM for T7 and 9.0 nM for qL2. The value for T7 is in close agreementwith the 3.2 μM K_(D) previously reported for the 2C scTCR [Manning etal. (1999) J. Exp. Med. 189:461-470]. These findings show that the yeastsystem, combined with CDR3α-directed mutagenesis, allows selection ofmutants with at least about 100-fold higher intrinsic binding affinitiesfor a particular pMHC ligand.

If the soluble scTCR has a high affinity for its pMHC ligand, then it isuseful, like antibodies, as a specific probe for cell-surface boundantigen. To confirm this, the soluble T7 and qL2 scTCR werebiotinylated, and the labeled-scTCR molecules were incubated withT2-L^(d) cells loaded with QL9, p2Ca, or MCMV (SEQ ID NO: 1). The qL2scTCR, but not the T7 scTCR, yielded easily detectable staining of theT2 cells that had been incubated with QL9 or p2Ca (FIGS. 4A-4B). It issignificant that p2Ca-upregulated cells were also readily detected byqL2 scTCR, as p2Ca is the naturally processed form of the peptiderecognized by the alloreactive clone 2C and it has an even loweraffinity than the QL9/L^(d) complex for the 2C TCR [Sykulev et al.(1994) supra].

The high affinity receptors described in our study were derived byvariation at the VJ junction, the same process that operates veryeffectively in vivo through gene rearrangements in T cells (Davis andBjorkman (1988) Nature 334:395-402). The fact that we could readilyisolate a diverse set of high affinity TCR in vitro indicates that thereis not a genetic or structural limitation to high affinity receptors.This supports the view that inherently low affinities of TCRs found invivo are due to a lack of selection for higher affinity and perhaps aselection for lower affinity (Sykulev et al. (1995) Proc. Natl. Acad.Sci. USA 92:11990-11992; Valitutti et al. (1995) Nature 375:148-151;Rabinowitz et al. (1996) Proc. Natl. Acad. Sci. USA 93:1401-1405). Inthis respect, the higher affinity TCRs of the present invention nowprovide the reagents for directly testing hypotheses about the effectsof affinity on T cell responses (Davis et al. (1998) Ann. Rev. Immunol.16:523-544; Sykulev et al. (1995) supra; Valitutti et al. (1995) supra;Rabinowitz et al. 1996) supra).

In summary, we have shown that T cell receptors, which represent a classof proteins as diverse as antibodies, can be engineered like antibodiesto yield high affinity, antigen-specific probes. Furthermore, a solubleversion of the high affinity receptor can directly detect specificpeptide/MHC complexes on cells. Thus, these engineered proteins areuseful as diagnostics, for tumor cells, for example. Soluble derivativesof the high affinity TCRs are useful or can be further engineered ashigh affinity, antigen-specific probes. The soluble TCR derivatives whenappropriately labeled (or bound by a detectable ligand for that solubleTCR) can serve as a probe for specific peptide/MCHC complexes on cells,for example, derived surfaces of tumor cells or other neoplastic cells,or antigens diagnostic of virus-infected cells or other diseased cells.Other applications for high affinity TCR cell bound proteins or solublederivatives include use in diagnosis or study of certain autoimmunediseases. Where a characteristic peptide/MHC or other marker surfaceantigen is known or can be identified, a high affinity, soluble TCR canbe isolated for specific binding to that cell surface moiety and used indiagnosis or in therapy. The high affinity TCR proteins, desirably thesoluble derivatives, can be used bound to cytotoxic agents astherapeutics in cancer treatment or other disorders where cells to bedesirably destroyed have a characteristic and specific cell surfacemoiety which is recognized by a high affinity TCR (desirably a solubleTCR protein). Similarly, a soluble high affinity TCR as described hereincan be coupled to an imaging agent and used to identify sites within thebody where tumor cells reside where the TCR specifically binds a tumorcell marker with high affinity and specificity. A high affinity TCRbound to the surface of a cell or tissue which has been inappropriatelytargeted for autoimmune destruction can reduce autoimmune tissuedestruction by cytotoxic lymphocytes by competing with those cytotoxiclymphocytes for binding to the cell surface of the targeted cells ortissue.

These results can also be considered in the context of an important,basic question in T cell responses. Are the low affinities previouslyobserved for T cell receptors due to the absence of somatic mutations ordue to in vivo selective pressures that act on the T cell? The highaffinity receptors described in our study were derived by variation atthe VJ junction, the same process that operates very effectively in Tcells [Davis and Bjorkman (1988) supra]. The fact that we could readilyisolate a diverse set of high affinity TCR in vitro indicates that thereis no structural or genetic limitation to high affinity receptors. Thissupports the view that inherently low affinities of TCRs found in vivoare due to a lack of selection for higher affinity and perhaps aselection for lower affinity [Sykulev et al. (1995) Proc. Natl. Acad.Sci. USA 92:11990-11992; Rabinowitz et al. (1996) supra]. In thisrespect, the higher affinity TCRs now provide the reagents for directlytesting hypotheses about the effects of affinity on T cell responses[Davis et al. (1998) supra; Sykulev et al. (1995) supra; Valitutti etal. (1995) supra; Rabinowitz et al. (1996) supra].

It will be appreciated by those of skill in the art that, due to thedegeneracy of the genetic code, numerous functionally equivalentnucleotide sequences encode the same amino acid sequence.

Additionally, those of skill in the art, through standard mutagenesistechniques, in conjunction with the antigen-finding activity assaysdescribed herein, can obtain altered TCR sequences and test them for theexpression of polypeptides having particular binding activity. Usefulmutagenesis techniques known in the art include, without limitation,oligonucleotide-directed mutagenesis, region-specific mutagenesis,linker-scanning mutagenesis, and site-directed mutagenesis by PCR [seee.g. Sambrook et al. (1989) and Ausubel et al. (1999)].

In obtaining variant TCR coding sequences, those of ordinary skill inthe art will recognize that TCR-derived proteins may be modified bycertain amino acid substitutions, additions, deletions, andpost-translational modifications, without loss or reduction ofbiological activity. In particular, it is well-known that conservativeamino acid substitutions, that is, substitution of one amino acid foranother amino acid of similar size, charge, polarity and conformation,are unlikely to significantly alter protein function. The 20 standardamino acids that are the constituents of proteins can be broadlycategorized into four groups of conservative amino acids as follows: thenonpolar (hydrophobic) group includes alanine, isoleucine, leucine,methionine, phenylalanine, proline, tryptophan and valine; the polar(uncharged, neutral) group includes asparagine, cysteine, glutamine,glycine, serine, threonine and tyrosine; the positively charged (basic)group contains arginine, histidine and lysine; and the negativelycharged (acidic) group contains aspartic acid and glutamic acid.Substitution in a protein of one amino acid for another within the samegroup is unlikely to have an adverse effect on the biological activityof the protein.

Homology between nucleotide sequences can be determined by DNAhybridization analysis, wherein the stability of the double-stranded DNAhybrid is dependent on the extent of base pairing that occurs.Conditions of high temperature and/or low salt content reduce thestability of the hybrid, and can be varied to prevent annealing ofsequences having less than a selected degree of homology. For instance,for sequences with about 55% G-C content, hybridization and washconditions of 40-50° C., 6×SSC (sodium chloride/sodium citrate buffer)and 0.1% SDS (sodium dodecyl sulfate) indicate about 60-70% homology,hybridization and wash conditions of 50-65° C., 1×SSC and 0.1% SDSindicate about 82-97% homology, and hybridization and wash conditions of52° C., 0.1×SSC and 0.1% SDS indicate about 99-100% homology. A widerange of computer programs for comparing nucleotide and amino acidsequences (and measuring the degree of homology) are also available, anda list providing sources of both commercially available and freesoftware is found in Ausubel et al. (1999). Readily available sequencecomparison and multiple sequence alignment algorithms are, respectively,the Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1997)and ClustalW programs. BLAST is available on the Internet atncbi.nlm.nih.gov and a version of ClustalW is available at 2.ebi.ac.uk.

Industrial strains of microorganisms (e.g., Aspergillus niger,Aspergillus ficuum, Aspergillus awamori, Aspergillus oryzae, Trichodermareesei, Mucor miehei, Kluyveromyces lactis, Pichia pastoris,Saccharomyces cerevisiae, Escherichia coli, Bacillus subtilis orBacillus licheniformis) or plant species (e.g., canola, soybean, corn,potato, barley, rye, wheat) may be used as host cells for therecombinant production of the TCR peptides. As the first step in theheterologous expression of a high affinity TCR protein or solubleprotein, an expression construct is assembled to include the TCR orsoluble TCR coding sequence and control sequences such as promoters,enhancers and terminators. Other sequences such as signal sequences andselectable markers may also be included. To achieve extracellularexpression of the scTCR, the expression construct may include asecretory signal sequence. The signal sequence is not included on theexpression construct if cytoplasmic expression is desired. The promoterand signal sequence are functional in the host cell and provide forexpression and secretion of the TCR or soluble TCR protein.Transcriptional terminators are included to ensure efficienttranscription. Ancillary sequences enhancing expression or proteinpurification may also be included in the expression construct.

Various promoters (transcriptional initiation regulatory region) may beused according to the invention. The selection of the appropriatepromoter is dependent upon the proposed expression host. Promoters fromheterologous sources may be used as long as they are functional in thechosen host.

Promoter selection is also dependent upon the desired efficiency andlevel of peptide or protein production. Inducible promoters such as tacare often employed in order to dramatically increase the level ofprotein expression in E. coli. Overexpression of proteins may be harmfulto the host cells. Consequently, host cell growth may be limited. Theuse of inducible promoter systems allows the host cells to be cultivatedto acceptable densities prior to induction of gene expression, therebyfacilitating higher product yields.

Various signal sequences may be used according to the invention. Asignal sequence which is homologous to the TCR coding sequence may beused. Alternatively, a signal sequence which has been selected ordesigned for efficient secretion and processing in the expression hostmay also be used. For example, suitable signal sequence/host cell pairsinclude the B. subtilis sacB signal sequence for secretion in B.subtilis, and the Saccharomyces cerevisiae α-mating factor or P.pastoris acid phosphatase phoI signal sequences for P. pastorissecretion. The signal sequence may be joined directly through thesequence encoding the signal peptidase cleavage site to the proteincoding sequence, or through a short nucleotide bridge consisting ofusually fewer than ten codons, where the bridge ensures correct readingframe of the downstream TCR sequence.

Elements for enhancing transcription and translation have beenidentified for eukaryotic protein expression systems. For example,positioning the cauliflower mosaic virus (CaMV) promoter 1000 bp oneither side of a heterologous promoter may elevate transcriptionallevels by 10- to 400-fold in plant cells. The expression constructshould also include the appropriate translational initiation sequences.Modification of the expression construct to include a Kozak consensussequence for proper translational initiation may increase the level oftranslation by 10 fold.

A selective marker is often employed, which may be part of theexpression construct or separate from it (e.g., carried by theexpression vector), so that the marker may integrate at a site differentfrom the gene of interest. Examples include markers that conferresistance to antibiotics (e.g., bla confers resistance to ampicillinfor E. coli host cells, nptII confers kanamycin resistance to a widevariety of prokaryotic and eukaryotic cells) or that permit the host togrow on minimal medium (e.g., HIS4 enables P. pastoris or His⁻ S.cerevisiae to grow in the absence of histidine). The selectable markerhas its own transcriptional and translational initiation and terminationregulatory regions to allow for independent expression of the marker. Ifantibiotic resistance is employed as a marker, the concentration of theantibiotic for selection will vary depending upon the antibiotic,generally ranging from 10 to 600 μg of the antibiotic/mL of medium.

The expression construct is assembled by employing known recombinant DNAtechniques (Sambrook et al., 1989; Ausubel et al., 1999). Restrictionenzyme digestion and ligation are the basic steps employed to join twofragments of DNA. The ends of the DNA fragment may require modificationprior to ligation, and this may be accomplished by filling in overhangs,deleting terminal portions of the fragment(s) with nucleases (e.g.,ExoIII), site directed mutagenesis, or by adding new base pairs by PCR.Polylinkers and adaptors may be employed to facilitate joining ofselected fragments. The expression construct is typically assembled instages employing rounds of restriction, ligation, and transformation ofE. coli. Numerous cloning vectors suitable for construction of theexpression construct are known in the art (λZAP and pBLUESCRIPT SK-1,Stratagene, LaJolla, Calif.; pET, Novagen Inc., Madison, Wis.—cited inAusubel et al., 1999) and the particular choice is not critical to theinvention. The selection of cloning vector will be influenced by thegene transfer system selected for introduction of the expressionconstruct into the host cell. At the end of each stage, the resultingconstruct may be analyzed by restriction, DNA sequence, hybridizationand PCR analyses.

The expression construct may be transformed into the host as the cloningvector construct, either linear or circular, or may be removed from thecloning vector and used as is or introduced onto a delivery vector. Thedelivery vector facilitates the introduction and maintenance of theexpression construct in the selected host cell type. The expressionconstruct is introduced into the host cells by any of a number of knowngene transfer systems (e.g., natural competence, chemically mediatedtransformation, protoplast transformation, electroporation, biolistictransformation, transfection, or conjugation) (Ausubel et al., 1999;Sambrook et al., 1989). The gene transfer system selected depends uponthe host cells and vector systems used.

For instance, the expression construct can be introduced into S.cerevisiae cells by protoplast transformation or electroporation.Electroporation of S. cerevisiae is readily accomplished, and yieldstransformation efficiencies comparable to spheroplast transformation.

Monoclonal or polyclonal antibodies, preferably monoclonal, specificallyreacting with a TCR protein at a site other than the ligand binding sitemay be made by methods known in the art. See, e.g., Harlow and Lane(1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratories;Goding (1986) Monoclonal Antibodies: Principles and Practice, 2d ed.,Academic Press, New York; and Ausubel et al. (1999) Current Protocols inMolecular Biology, John Wiley & Sons, Inc., New York.

High affinity TCR proteins in cell-bound or soluble form which arespecific for a particular pMHC are useful, for example, as diagnosticprobes for screening biological samples (such as cells, tissue samples,biopsy material, bodily fluids and the like) or for detecting thepresence of the cognate pMHC in a test sample. Frequently, the highaffinity TCR proteins are labeled by joining, either covalently ornoncovalently, a substance which provides a detectable signal. Suitablelabels include but are not limited to radionuclides, enzymes,substrates, cofactors, inhibitors, fluorescent agents, chemiluminescentagents, magnetic particles and the like. Additionally the TCR proteincan be coupled to a ligand for a second binding molecules: for example,the TCR protein can be biotinylated. Detection of the TCR bound to atarget cell or molecule can then be effected by binding of a detectablestreptavidin (a streptavidin to which a fluorescent, radioactive,chemiluminescent, or other detectable molecule is attached or to whichan enzyme for which there is a chromophoric substrate available). UnitedStates patents describing the use of such labels and/or toxic compoundsto be covalently bound to the scTCR protein include but are not limitedto U.S. Pat. Nos. 3,817,837; 3,850,752; 3,927,193; 3,939,350; 3,996,345;4,277,437; 4,275,149; 4,331,647; 4,348,376; 4,361,544; 4,468,457;4,444,744; 4,640,561; 4,366,241; RE 35,500; 5,299,253; 5,101,827;5,059,413. Labeled TCR proteins can be detected using a monitoringdevice or method appropriate to the label used. Fluorescence microscopyor fluorescence activated cell sorting can be used where the label is afluorescent moiety, and where the label is a radionuclide, gammacounting, autoradiography or liquid scintillation counting, for example,can be used with the proviso that the method is appropriate to thesample being analyzed and the radionuclide used. In addition, there canbe secondary detection molecules or particle employed where there is adetectable molecule or particle which recognized the portion of the TCRprotein which is not part of the binding site for the cognate pMHCligand or other ligand in the absence of a MHC component as notedherein. The art knows useful compounds for diagnostic imaging in situ;see, e.g., U.S. Pat. Nos. 5,101,827; 5,059,413. Radionuclides useful fortherapy and/or imaging in vivo include ¹¹¹Indium, ⁹⁷Rubidium, ¹²⁵Iodine,¹³¹Iodine, ¹²³Iodine, ⁶⁷Gallium, ⁹⁹Technetium. Toxins include diphtheriatoxin, ricin and castor bean toxin, among others, with the proviso thatonce the TCR-toxin complex is bound to the cell, the toxic moiety isinternalized so that it can exert its cytotoxic effect. Immunotoxintechnology is well known to the art, and suitable toxic moleculesinclude, without limitation, chemotherapeutic drugs such as vindesine,antifolates, e.g. methotrexate, cisplatin, mitomycin, anthrocyclinessuch as daunomycin, daunorubicin or adriamycin, and cytotoxic proteinssuch as ribosome inactivating proteins (e.g., diphtheria toxin, pokeweedantiviral protein, abrin, ricin, pseudomonas exotoxin A or theirrecombinant derivatives. See, generally, e.g., Olsnes and Pihl (1982)Pharmac. Ther. 25:355-381 and Monoclonal Antibodies for Cancer Detectionand Therapy, Eds. Baldwin and Byers, pp. 159-179, Academic Press, 1985.

High affinity TCR proteins specific for a particular pMHC ligand areuseful in diagnosing animals, including humans believed to be sufferingfrom a disease associated with the particular pMHC. The sc TCR moleculesof the present invention are useful for detecting essentially anyantigen, including but not limited to, those associated with aneoplastic condition, an abnormal protein, or an infection orinfestation with a bacterium, a fungus, a virus, a protozoan, a yeast, anematode or other parasite. The high affinity sc TCR proteins can alsobe used in the diagnosis of certain genetic disorders in which there isan abnormal protein produced. Exemplary applications for these highaffinity proteins is in the treatment of autoimmune diseases in whichthere is a known pMHC. Type I diabetes is relatively well characterizedwith respect to the autoantigens which attract immune destruction.Multiple sclerosis, celiac disease, inflammatory bowel disease, Crohn'sdisease and rheumatoid arthritis are additional candidate diseases forsuch application. High affinity TCR (soluble) proteins with bindingspecificity for the p/MHC complex on the surface of cells or tissueswhich are improperly targeted for autoimmune destruction can serve asantagonists of the autoimmune destruction by competing for binding tothe target cells by cytotoxic lymphocytes. By contrast, high affinityTCR proteins, desirably soluble single chain TCR proteins, whichspecifically bind to an antigen or to a p/MHC on the surface of a cellfor which destruction is beneficial, can be coupled to toxic compounds(e.g., toxins or radionuclides) so that binding to the target cellresults in subsequent binding and destruction by cytotoxic lymphocytes.The cell targeted for destruction can be a neoplastic cell (such as atumor cell), a cell infected with a virus, bacterium or protozoan orother disease-causing agent or parasite, or it can be a bacterium,yeast, fungus, protozoan or other undesirable cell. Such high affinitysc TCR proteins can be obtained by the methods described herein andsubsequently used for screening for a particular ligand of interest.

The high affinity TCR compositions can be formulated by any of the meansknown in the art. They can be typically prepared as injectables,especially for intravenous, intraperitoneal or synovial administration(with the route determined by the particular disease) or as formulationsfor intranasal or oral administration, either as liquid solutions orsuspensions. Solid forms suitable for solution in, or suspension in,liquid prior to injection or other administration may also be prepared.The preparation may also, for example, be emulsified, or theprotein(s)/peptide(s) encapsulated in liposomes.

The active ingredients are often mixed with excipients or carriers whichare pharmaceutically acceptable and compatible with the activeingredient. Suitable excipients include but are not limited to water,saline, dextrose, glycerol, ethanol, or the like and combinationsthereof. The concentration of the high affinity TCR protein ininjectable, aerosol or nasal formulations is usually in the range of0.05 to 5 mg/ml. The selection of the particular effective dosages isknown and performed without undue experimentation by one of ordinaryskill in the art. Similar dosages can be administered to other mucosalsurfaces.

In addition, if desired, vaccines may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,and/or adjuvants which enhance the effectiveness of the vaccine.Examples of adjuvants which may be effective include but are not limitedto: aluminum hydroxide; N-acetyl-muramyl-L-threonyl-D-isoglutamine(thr-MDP); N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637,referred to as nor-MDP);N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE); and RIBI, which contains threecomponents extracted from bacteria: monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween80 emulsion. Such additional formulations and modes of administration asare known in the art may also be used.

The high affinity TCR proteins of the present invention and/orpMHC-binding fragments having primary structure similar (more than 90%identity) to the high affinity TCR proteins and which maintain the highaffinity for the cognate ligand may be formulated into vaccines asneutral or salt forms. Pharmaceutically acceptable salts include but arenot limited to the acid addition salts (formed with free amino groups ofthe peptide) which are formed with inorganic acids, e.g., hydrochloricacid or phosphoric acids; and organic acids, e.g., acetic, oxalic,tartaric, or maleic acid. Salts formed with the free carboxyl groups mayalso be derived from inorganic bases, e.g., sodium, potassium, ammonium,calcium, or ferric hydroxides, and organic bases, e.g., isopropylamine,trimethylamine, 2-ethylamino-ethanol, histidine, and procaine.

High affinity TCR proteins for therapeutic use, e.g., those conjugatedto cytotoxic compounds are administered in a manner compatible with thedosage formulation, and in such amount and manner as areprophylactically and/or therapeutically effective, according to what isknown to the art. The quantity to be administered, which is generally inthe range of about 100 to 20,000 μg of protein per dose, more generallyin the range of about 1000 to 10,000 μg of protein per dose. Similarcompositions can be administered in similar ways using labeled highaffinity TCR proteins for use in imaging, for example, to detect tissueunder autoimmune attack and containing the cognate pMHCs or to detectcancer cells bearing a cognate pMHC on their surfaces. Precise amountsof the active ingredient required to be administered may depend on thejudgment of the physician or veterinarian and may be peculiar to eachindividual, but such a determination is within the skill of such apractitioner.

The vaccine or other immunogenic composition may be given in a singledose; two dose schedule, for example two to eight weeks apart; or amultiple dose schedule. A multiple dose schedule is one in which aprimary course of vaccination may include 1 to 10 or more separatedoses, followed by other doses administered at subsequent time intervalsas required to maintain and/or reinforce the immune response, e.g., at 1to 4 months for a second dose, and if needed, a subsequent dose(s) afterseveral months. Humans (or other animals) immunized with theretrovirus-like particles of the present invention are protected frominfection by the cognate retrovirus.

Standard techniques for cloning, DNA isolation, amplification andpurification, for enzymatic reactions involving DNA ligase, DNApolymerase, restriction endonucleases and the like, and variousseparation techniques are those known and commonly employed by thoseskilled in the art. A number of standard techniques are described inSambrook et al. (1989) Molecular Cloning, Second Edition, Cold SpringHarbor Laboratory, Plainview, N.Y.; Maniatis et al. (1982) MolecularCloning, Cold Spring Harbor Laboratory, Plainview, N.Y.; Wu (ed.) (1993)Meth. Enzymol. 218, Part I; Wu (ed.) (1979) Meth Enzymol. 68; Wu et al.(eds.) (1983) Meth. Enzymol. 100 and 101; Grossman and Moldave (eds.)Meth. Enzymol. 65; Miller (ed.) (1972) Experiments in MolecularGenetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Oldand Primrose (1981) Principles of Gene Manipulation, University ofCalifornia Press, Berkeley; Schleif and Wensink (1982) Practical Methodsin Molecular Biology; Glover (ed.) (1985) DNA Cloning Vol. I and II, IRLPress, Oxford, UK; Hames and Higgins (eds.) (1985) Nucleic AcidHybridization, IRL Press, Oxford, UK; and Setlow and Hollaender (1979)Genetic Engineering: Principles and Methods, Vols. 1-4, Plenum Press,New York. Abbreviations and nomenclature, where employed, are deemedstandard in the field and commonly used in professional journals such asthose cited herein.

All references cited in the present application are incorporated byreference herein to supplement the disclosure and experimentalprocedures provided in the present Specification to the extent thatthere is no inconsistency with the present disclosure.

The following examples are provided for illustrative purposes, and arenot intended to limit the scope of the invention as claimed herein. Anyvariations in the exemplified articles and/or methods which occur to theskilled artisan are intended to fall within the scope of the presentinvention.

EXAMPLES

General methods for making high affinity TCRs are given in U.S. patentapplication Ser. No. 09/009,388, filed Jan. 20, 1998, and WO99/36569,filed Jan. 20, 1999, which are hereby incorporated by reference to theextent not inconsistent with the disclosure herewith.

Example 1 Library Construction

The 2C scTCR used as the scaffold for directed evolution (T7) containedsix mutations (βG17E, βG42E βL81S, αL43P, αW82R, and αI1118N) that havebeen shown to increase the stability of the TCR but still allow pMHCbinding [see, e.g., Shusta, E. V. et al. (1999) J. Mol. Biol. 292,949-956].

Mutagenic PCR of the T7 scTCR VαCDR3 was performed using anAGA2-specific upstream primer (GGCAGCCCCATAAACACACAGTAT (SEQ ID NO:3))and a degenerate downstream primerCTTTTGTGCCGGATCCAAATGTCAG(SNN)₅GCTCACAGCACAGAAGTACACGGCCGA GTCGCTC (SEQID NO:4). Underlined bases indicate the positions of silent mutationsintroducing unique BamHI and EagI restriction sites. The purified PCRproduct was digested with NdeI and BamHI and ligated to NdeI-BamHIdigested T7/pCT302 [Boder and Wittrup (1997) supra; Kieke et al. (1999)supra; Shusta et al. (1999) supra]. The ligation mixture was transformedinto DH10B electro-competent E. coli (Gibco BRI, Gaithersburg, Md.), andtransformants were pooled into 250 ml LB supplemented with ampicillin at100 μg/ml and grown overnight at 37° C. Plasmid DNA was transformed intothe yeast, (Saccharomyces cerevisiae) strain EBY100 by the method ofGietz and Schiestl [Geitz et al. (1995) Yeast 11:355-360].

Example 2 Cell Sorting

The yeast library [Shusta et al. (1999) Curr. Opin. Biotechnol.10:117-122] was grown in SD-CAA (2% dextrose, 0.67% yeast nitrogen base,1% casamino acids (Difco, Livonia, Mich.)) at 30° C. to an OD₆₀₀=4.0. Toinduce surface scTCR expression, yeast were pelleted by centrifugation,resuspended to an OD₆₀₀=1.0 in SG-CAA (2% galactose, 0.67% yeastnitrogen base, 1% casamino acids), and incubated at 20° C. for about 24hr. In general, about 10⁷ cells/tube were incubated on ice for 1 hr with50 μl of QL9/L^(d)/IgG dimers [Dal Porto et al. (1993) supra] diluted inphosphate buffered saline, pH 7.4 supplemented with 0.5 mg/ml BSA(PBS-BSA). After incubation, cells were washed and labeled for 30 minwith FITC-conjugated goat anti-mouse IgG F(ab⁷)₂ (Kirkegaard & Perry,Gaithersburg, Md.) in PBS-BSA. Yeast were then washed and resuspended inPBS-BSA immediately prior to sorting. Cells exhibiting the highestfluorescence were isolated by FACS sorting with a Coulter 753 bench.After isolation, sorted cells were expanded in SD-CAA and induced inSG-CAA for subsequent rounds of selection. A total of four sequentialsorts were performed. The concentrations of QL9/L^(d)/IgG dimers usedfor staining were 50 μg/ml for sorts 1-3 and 0.5 μg/ml for the finalsort. The percentages of total cells isolated from each sort were 5.55,2.68, 2.56, and 0.58%, respectively. Aliquots of sorts 3 and 4 wereplated on SD-CAA to isolate individual clones which were analyzed byflow cytometry using a Coulter Epics XL instrument.

Example 3 Soluble scTCR Production

The T7 and qL2 open reading frames were excised from pCT302 NheI-XhoIand ligated into NheI-XhoI digested pRSGALT, a yeast expression plasmid[Shusta et al. (1999) supra]. Ligated plasmids were transformed intoDH10B electro-competent E. coli (Gibco BRL). Plasmid DNA was isolatedfrom bacterial cultures and transformed into Saccharomyces cerevisiaeBJ5464 (α ura3-52 trp1 leu2 1 his3 200 pep4::HIS3 prb1 1.6R can1 GAL)[Shusta et al. (1999) supra]. Yeast clones were grown in one literSD-CAA/Trp (20 mg/L tryptophan) for 48 hr at 30° C. To induce scTCRsecretion, cells were pelleted by centrifugation at 4000×g, resuspendedin one liter SG-CAA/Trp supplemented with 1 mg/ml BSA, and incubated for72 hr at 20° C. Culture supernatants were harvested by centrifugation at4000×g, concentrated to about 50 ml, and dialyzed against PBS, pH 8.0.The 6His-tagged scTCRs were purified by native nickel affinitychromatography (Ni-NTA Superflow, Qiagen, Valencia, Calif.; 5 mM and 20mM imidazole, pH 8.0 wash; 250 mM imidazole elution) [Shusta et al.(1999) supra].

Example 4 Cell-Binding Assays

The binding of soluble scTCRs to QL9/L^(d) was monitored in acompetition format as described previously [Manning et al. (1998) supra;Sykulev et al. (1994) supra]. Peptide-upregulated T2-L^(d) cells(3×10⁵/well) were incubated for one hour on ice in the presence of¹²⁵I-labeled anti-L^(d) Fabs (30-5-7) and various concentrations ofscTCRs. Bound and unbound ¹²⁵I 30-5-7 Fabs were separated bycentrifugation through olive oil/dibutyl phthalate. Inhibition curveswere constructed to determine inhibitor concentrations yielding 50% ofmaximal inhibition. Dissociation constants were calculated using theformula of Cheng and Pursoff [Cheng (1973) Biochem. Pharm.22:3099-3108]. To monitor direct binding of scTCRs to cell-bound pMHC,peptide-upregulated T2-L^(d) cells (5×10⁵/tube) were incubated for 40min on ice with biotinylated soluble scTCRs followed by staining for 30min with streptavidin-phycoerythrin (PharMingen, San Diego, Calif.).Cellular fluorescence was detected by flow cytometry.

Example 5 Identification of High Affinity TCRs that are Specific for aDifferent Peptide and a Different MHC Molecule (K^(b))

Using the same library of yeast-displayed mutants of the CDR3α region ofthe TCR, it was possible to select for higher affinity TCRs that arespecific for yet a different peptide bound to a different MHC molecule.In this case the peptide called SIYR (SIYRYYGL (SEQ ID NO:5)) was boundto the MHC molecule called K^(b), and this ligand complex was used influorescent form to select by flow cytometry. Sixteen clones expressinghigh affinity TCR were sequenced, each showing a different sequence inthe CDR3α region (FIG. 11).

As an example of the specificity of these TCRs, the mutant 3SQ2 wasstained with various agents, including the secondary reagent alone(SA:PE), the anti-Vβ antibody F23.2, and three peptide/K^(b) complexes(OVA/K^(b), dEV8/K^(b), and SIYR/K^(b)). As shown in FIG. 5, only thepMHC(SIYR/K^(b)) used in the original selection had sufficient affinityto bind to the mutant TCR. Wild-type TCR did not bind the SIYR/K^(b)ligand at any concentration tested (data not shown).

The mutant TCR 3SQ2 was also expressed as a soluble protein in the yeastsecretion system and tested after biotinylation for its ability to binddirectly to pMHC on the surface of tumor cells. As shown in FIG. 6, thelabeled 3SQ2 bound very well to tumor cells that expressed only theappropriate peptide SIYR. The staining was nearly as strong as the highaffinity anti-Kb monoclonal antibody B8.24.3, that binds to any K^(b)molecule (FIG. 6), regardless of the peptide present.

Example 6 Identification of High Affinity TCRs that are Specific for aDifferent Peptide Bound to the Same MHC Molecule (K^(b))

To determine if the same TCR scaffold could be used to isolate higheraffinity forms against yet a different peptide bound to the same MHC, wescreened the TCR CDR3α library with the peptide called dEV8 (EQYKFYSV(SEQ ID NO:6)), bound to K^(b). After several sorts by flow cytometrywith the biotinylated dEV8/K^(b) ligand (followed byphycoerythrin-streptavidin, PE-SA), there was a significant enrichmentof yeast cells that bound to the dEV8/Kb (as indicated by PE levels inFIG. 7).

Six of the clones that were isolated by selection with dEV8/K^(b) weresequenced and the CDR3 sequences all differed (FIG. 12). These sequenceswere similar in sequence, but different from, those isolated byselection with SIYR/K^(b) (two examples, 3SQ2 and 3SQ5, are also shownin FIG. 12. It can be concluded that it is possible to isolate higheraffinity TCRs against different antigens, even using the same TCRlibrary of mutants.

To prove the antigen specificity of the isolated clones, one of thedEV8/K^(b) selected clones (4d1) was examined with a panel of differentantibodies and ligands (FIG. 6). As expected, this TCR reacted with thethree appropriate antibodies (anti-Vβ8 antibody F23.2, anti-HA tagantibody, and anti-His tag antibody) and the dEV8/K^(b) antigen, but notwith another antigen, OVA/K^(b). Wild type TCR did not bind to eitherpeptide/K^(b) complex (data not shown). Thus, the high affinity TCR wasspecific for the selected antigen.

Example 7 Identification of High Affinity TCRs by Creating a Random TCRLibrary in a Different Region of the TCR (Complementarity-DeterminingRegion 3 of the β Chain)

The examples described above used a library of TCR that were mutatedwithin a region of the α chain called CDR3. In order to show that otherregions of the TCR could also be mutated to yield higher affinity TCR, alibrary of random mutants within five contiguous amino acid residues ofthe CDR3 region of the β chain was produced, using the qL2 TCR mutant asthe starting material. This library was then selected with the QL9/L^(d)ligand at concentrations below that detected with the qL2 mutant. Fiveyeast clones, selected by flow cytometry, were sequenced and each showeda different nucleotide and amino acid sequence (FIG. 8). There wasremarkable conservation of sequence within the five amino acid regionthat was mutated, suggesting that this sequence motif has been optimizedfor high affinity. We conclude that it is possible to mutate differentregions of the TCR to yield derivatives having higher affinity for aparticular pMHC.

Although the description above contains many specificities, these shouldnot be construed to limit the scope of the invention but as merelyproviding illustrations of some of the presently-preferred embodimentsof this invention. For example, ligands other than those specificallyillustrated may be used. Thus the scope of the invention should bedetermined by the appended claims and their legal equivalents, ratherthan by the examples given. All references cited herein are incorporatedto the extent not inconsistent with the disclosure herewith.

1. A method for cloning the gene for a high affinity TCR mutant into asystem that allows expression of the mutant on the surface of T cellscomprising: mutating TCRs to create high affinity TCR mutants; cloningsaid TCR mutants into a vector; transfecting the vector into T cells;expressing the high affinity TCR mutant on the surface of T cells. 2.The method of claim 1, further comprising: selecting those T cells thatare activated by a peptide/MHC ligand more than the wild type.
 3. Themethod of claim 1, wherein the transfected/infected T cells are used forrecognition of selected peptide-bearing MHC cells.
 4. T cells made bythe method of claim
 1. 5. A method for cloning the gene for a highaffinity TCR mutant into a system that allows expression of the mutanton the surface of T cells comprising the steps of: mutating TCRs tocreate high affinity TCR mutants which exhibit a dissociation constantfor their cognate ligand of at least about 10⁷ M⁻¹; cloning said TCRmutants into a vector; transfecting the vector into T cells; andexpressing the high affinity TCR mutant on the surface of T cells. 6.The method of claim 5, wherein the transfected T cells are used forrecognition of selected peptide-bearing MHC cells.
 7. The method ofclaim 5 wherein the high affinity TCR mutants carry one or moremutations in a CDR.
 8. The method of claim 7 wherein the high affinityTCR mutants carry one or more mutations in CDR3α or CDR3β.
 9. A methodfor cloning the gene for a high affinity TCR mutant into a system thatallows expression of the mutant on the surface of T cells comprising thesteps of: mutating TCRs to create high affinity TCR mutants carrying oneor more mutations in a CDR; cloning said TCR mutants into a vector;transfecting the vector into T cells; and expressing the high affinityTCR mutant on the surface of T cells.
 10. The method of claim 9, whereinthe transfected T cells are used for recognition of selectedpeptide-bearing MHC cells.
 11. The method of claim 9 wherein the highaffinity TCR mutants carry one or more mutations in CDR3α or CDR3β. 12.T cells made by the methods of claim
 5. 13. A DNA sequence encoding amutant high affinity TCR exhibiting a dissociation constant of greaterthan about 10⁷ M⁻¹ for its cognate ligand.
 14. The DNA sequence of claim13 wherein the TCR mutant exhibits a dissociation constant between about10⁷ and 10¹⁰ M⁻¹ for its cognate ligand.
 15. The DNA sequence of claim13 wherein the TCR mutant carries one or more mutations in a CDR. 16.The DNA sequence of claim 15 wherein the TCR mutant carries one or moremutations in CDR3α or CDR3β.
 17. A DNA sequence encoding a mutant highaffinity TCR carrying one or more mutations in CDR.
 18. The DNA sequenceof claim 17 carrying one or mutations in CDR3α or CDR3β.
 19. T cellsmade by the methods of claim 9.